Field of the Invention
This invention relates to a nucleic acid encoding a functional module or domain of a particular peptidoglycan hydrolase, the streptococcal phage λSa2 endolysin, a protein which digests the peptidoglycan cell wall of streptococcal species with near species-specificity. Optimal Ca++ and NaCl concentrations and pH have been determined for native λSa2 endolysin and for multiple truncations of λSa2 endolysin. The invention further relates to lysis of untreated, live streptococcal bacteria observed as a result of native and truncated λSa2 lysis activity and enhancement of that activity on streptococcal bacteria as well as staphylococcal bacteria when the nucleic acid encoding the functional endopeptidase domain is further modified with one or more copies of endogenous phage cell wall-binding domains, i.e., Cpl-7 domains or with the SH3b cell wall-binding domain from the bacteriocin lysostaphin. The invention further relates to compositions and methods of treating diseases caused by the bacteria for which the λSa2 endopeptidase and modified λSa2 endopeptidase are specific, namely, streptococcal diseases, and in addition, compositions and methods comprising λSa2 endopeptidase for treating staphylococcal-associated diseases, including methicillin-resistant Staphylococcus aureus (MRSA).
Description of the Relevant Art
Streptococci are notorious pathogens in animals and man, with S. pneumoniae being most widely recognized for the pneumonia it causes (2007. Morb. Mortal. Wkly. Rep. 56:1077-1080). Other streptococci are renowned, for example, for human diseases such as group A streptococcus (GAS) pharyngo-tonsillitis (Passali et al. 2007. Acta Otorhinolaryngol. Ital. 27:27-32), group C streptococcus (GCS)-associated wound infections, otitis media, purulent pharyngitis, and streptococcal toxic shock syndrome (Davies et al. 2007. Clin. Infect. Dis. 44:1442-1454; Salata et al. 1989. Medicine (Baltimore) 68: 225-239), and group G streptococcus (GGS)-associated bacteremia (Sylvetsky et al. 2002. Am. J. Med. 112:622-626). Similarly, for the animal diseases, examples are: GCS Streptococcus equi subsp. zooepidemicus of haemorrhagic pneumonia in dogs (Kim et al. 2007. Vet. Rec. 161: 528-530), and (GES) group E streptococcal lymphadenitis of swine (Wessman, G. E. 1986. Vet. Microbiol. 12: 297-328). Streptococci having the ability to infect both humans and livestock occur as well, as is seen in group B streptococcus (GBS) mastitis (Wilson et al. 1997. J. Dairy Sci. 80: 2592-2598), perinatal GBS disease of infants (2007. Morb. Mortal. Wkly. Rep. 56:701-705), and in S. suis causing septicemia, meningitis, endocarditis and arthritis in pigs and causing fever, malaise, nausea and vomiting, followed by nervous symptoms, subcutaneous hemorrhage, septic shock and coma in humans (Gottschalk et al. 2007. Anim. Health Res. Rev. 8:29-45). The streptococci are following the trend of all antibiotic-treated pathogens with high levels of antibiotic resistance development being noted by clinicians (2007. Moth. Mortal. Wkly. Rep. 56:1077-1080; Niederman, M. S. 2007. Chest. 131:1205-1215; Passali et al., supra) and veterinarians (Lloyd, D. H. 2007. Clin. Infect. Dis. 45 (Suppl. 2):S148-52; Mathew et al. 2007. Foodbome Patholog. Dis. 4:115-133).
In this age of increasing resistance to antibiotics, efforts to find novel antimicrobials are turning to bactericidal proteins of bacterial and/or phage origin (Hermoso et al. 2007. Curr. Opin. Microbiol. 10: 461-472). Many Gram positive bacteriophage endolysins have been tested and shown to be efficacious when exposed externally to host-related pathogens (lysis from without or exolysis) (for recent reviews, see Loessner, M. J. 2005. Curr. Opin. Microbiol. 8:480-487; Fischetti, V. A. 2005. Trends Microbiol. 13: 491-496). In fact, lysostaphin (a bacteriocin secreted by Staphylococcus simulans to kill S. aureus) recently achieved higher efficacy than vancomycin against clinical isolates of multi-drug resistant Staphylococcus aureus (Yang et al. 2007. J. Med. Microbiol. 56: 71-76).
In the search for new antimicrobials, it has been deemed essential by FDA, CDC, USDA and other US government agencies in an interagency forum to develop agents that avoid resistance Retrieved from the Internet: <URL: cdc.gov/drugresistance/actionplan/2005report/index.htm. In support of this federal objective, the near-species specificity of bacteriophage endolysins is anticipated to help avoid resistance development among the non-related commensal bacteria that are inadvertently exposed to antimicrobials, something that occurs routinely during antibiotic treatment episodes. Furthermore, for the few bacteriophage endolysins tested, no resistant strain development has been identified (reviewed in Fischetti, supra). In addition, the efficacy of peptidoglycan hydrolases as an antimicrobial agent has been demonstrated in animal models of human disease (Cheng at al. 2005. Antimicrob. Agents Chemother. 49:111-117; Entenza et al. 2005. Antimicrob. Agents Chemother. 49:4789-4792; Rashel et al. 2007. J. Infect. Dis. 196:1237-1247) as well as in animal disease applications e.g. transgenic cattle with mammary expression of lysostaphin have been shown to be resistant to S. aureus-induced mastitis (Wall et al. 2005. Nat. Biotechnol. 23: 445-451).
Most Gram Positive lysins (bacterial or, phage origin) have an N-terminal lytic domain and a C-terminal cell wall-binding domain. The lytic domain of the phage endolysin can have any of three functions: an amidase activity, an endopeptidase, or a lysozyme-like glycosidase activity. The cell wall bonds sensitive to amidase (N-acetylmuramyl-L-alanine) and glycosidase lysin activities (β-1,4-linked N-acetylglucosamine and N-acetyl muramic acid residues) are conserved in the peptidoglycan of nearly all bacterial species, while the amino acid sequence of the peptide portion of peptidoglycan that is sensitive to endopeptidase activity can vary between species and genera (Schleifer and Kandler. 1972. Bacteria Rev. 36: 407-477).
The stem peptide region of peptidoglycan is often conserved across multiple species, within any given genus. Species-specificity in peptidoglycan structure usually occurs in the interpeptide bridge (Schleifer and Kandler, supra). The result is that phage lysins that recognize and cleave bonds in the regions of the peptidoglycan that are conserved within a genus will often lyse a much broader range of species than just the host range of their phage of origin. For example, the streptococcal B30 bacteriophage is hosted by a relatively small subgroup of type III GBS, but the B30 phage lysin can kill streptococci of Lancefield groups A, B, C, E, and G, with the rate of lysis for groups A, C and G being much higher than the host GBS (Pritchard at al. 2004. Microbiology 150: 2079-2087).
Species-specificity of these lysins can originate in part from both the lytic and cell wall-binding domains. The species-specificity of lysin endopeptidase domains can easily be ascribed to the differences in amino acid sequences of the peptidoglycan. This has been demonstrated experimentally, with digestion of short synthetic peptides that mimic the bonds recognized by the endopeptidase domain in question (Pritchard et al. 2007. Appl. Environ. Microbiol. 73: 7150-7154). The bonds cleaved by amidase and glycosidase activities of lysins are so highly conserved among all bacterial species that it is difficult to imagine how these structures themselves confer specificity to the lysins. However, it is known that O-acetylation at the C6-OH of the N-acetylmuramic acid can affect lysozyme's ability to digest the sugar backbone (Bera et al. 2006. Infect. Immun. 74: 4598-4604), with only a few enzymes known with the ability to cleave this O-acetylated sugar backbone. (Pritchard et al. 2007, supra; Yokogawa et al. 1974. Antimicrob. Agents Chemother. 6:156-165). That being said, there are still other largely undefined factors that contribute to species-specificity of the lysins. A hint at one source of this specificity is apparent when it was reported that the B30 endolysin with an inactivated endopeptidase domain resulting from a site-directed mutation and an active Acm lysozyme-like domain could digest the prepared peptidoglycan of GBS (Pritchard et al. 2004, supra), but the same lysin could not lyse GBS as a growing cell (Donovan et al. 2006b. Appl. Environ. Microbiol. 72:5108-5112). These results suggest that non-peptidoglycan factors (i.e. present on growing cells, but not prepared peptidoglycan) can alter peptidoglycan sensitivity to the lysin. There are numerous other structures on the Gram Positive cell wall that might participate in defining peptidoglycan hydrolase specificity, e.g. teichoic acid, capsule, poly-N-acetylglucosamine. Their potential role in endolysin specificity is undefined.
The C-terminal cell wall-binding domain of peptidoglycan hydrolase enzymes is known to effect cell-type specificity. The SH3b domain of the staphylococcal lytic enzyme ALE-1 (which is virtually identical to the SH3b domain of lysostaphin) has been examined via both mutagenesis and crystallography (Lu et al. 2006b. J. Biol. Chem. 281: 549-558). This and other studies (Grundling and Schneewind. 2006. J. Bacterial. 188: 2463-2472) have indicated that the SH3b domain recognizes the penta-glycine interpeptide bridge of staphylococcal peptidoglycan. Prior studies have indicated that lysostaphin requires the C-terminal 92 amino acids, including the SH3b domain, for cell-type specificity (Baba and Schneewind. 1996. EMBO J. 15:4789-4797). Similarly, the SH3b domain of the PlyB endolysin from a Bacillus anthrasis phage is required for high levels of activity (Porter et al. 2007. J. Mol. Biol. 366: 540-550). Essential choline-binding domains that are specific for streptococcal cell walls have also been described (Garcia et al. 1990. Gene 86: 81-88; Sanchez-Puelles et al. 1990. Gene 89: 69-75) with the CPL-1 lysin harboring one such domain having been recently crystallized (Perez-Dorado at al. 2007. J. Biol. Chem. 282:24990-24999). Again, this endolysin demonstrates an absolute need for the choline-binding domain for lytic activity (see a recent review of those lysins which have been crystallized with cell wall-binding domains, Koehl et al. 2004. Antimicrob. Agents Chemother. 48: 3749-3757).
Despite an absolute requirement of a cell wall-binding domain for the full lytic activity of some lysins, numerous examples of higher activity from truncated endolysins lacking their SH3b domains have been reported, e.g., staphylococcal phage Twort (Loessner et al. 1998. FEMS Microbiol. Lett. 162:265-274) and phiWMY endolysins (Yokoi et al. 2005. Gene 351: 97-108), streptococcal phage endolysin PlyGBS (Cheng and Fischetti. 2007. Appl. Microbiol. Biotechnol. 74:1284-1291), and streptococcal B30 endolysin (Donovan at al. 2006. Appl. Environ. Microbiol. 72(7):5108-5112; Donovan at al. 2006. Appl. Environ. Microbiol. 72(4):2988-96); Bacillus anthrasis Lambda phage endolysin PlyL lambda (Low at al. 2005. J. Biol. Chem. 280:35433-35439). This apparent inconsistency is further complicated by the fact that some lytic domains are not only more active, but maintain their cell type specificity in the absence of their SH3b cell wall-binding domain (Donovan et al. 2006b, supra). Still other domains do not show any activity toward their growing host when exposed externally (Donovan et al. 2007, supra) despite evidence that the domain is active on prepared peptidoglycan (Pritchard et al. 2004, supra). These inconsistent results dictate that a thorough analysis is needed of candidate peptidoglycan hydrolase antimicrobials to identify the most active domains and the constructs required to achieve lytic activity.
To counter the rise of drug resistant pathogenic bacteria, there is a need for new specific antimicrobial treatments. Reagents shown to be very specific for the genera, species or substrains of concern would give better effective control of economically important diseases and therefore are ideal candidates for therapeutic treatments. In this study we have identified the domains necessary for lysis from without (exolysis) for the Lambda Sa2 endolysin (λSa2), identified constructs that enhance the native enzyme activity level against the target species, (streptococcal), and extended the list of pathogen species known to be lysed by the enzyme.